WO2010112678A1

WO2010112678A1 - Regenerative matrix comprising cells activated by nemosis and/or factors released from such cells

Info

Publication number: WO2010112678A1
Application number: PCT/FI2010/050256
Authority: WO
Inventors: Jozef Bizik; Ari Harjula; Esko Kankuri
Original assignee: Helsingin Yliopisto
Priority date: 2009-04-01
Filing date: 2010-03-31
Publication date: 2010-10-07
Also published as: FI20095355A0

Abstract

The present invention relates to a product aimed at wound healing. The product of the invention comprises a specific set of stimulating factors secreted by biologically activated human cells combined with an active carrier matrix. Alternatively, such activated cells as such or a combination of such cells and the secreted factors may be added into the matrix. The uses are directed at healing and regeneration of injured tissues and organs.

Description

Regenerative matrix comprising cells activated by nemosis and/or factors released from such cells

The skin, consisting basically of the keratinocyte-rich epidermis and of the relatively acel- hilar deeper supporting connective tissue layer (Figure 6) together with sweat and sebaceous glands and hair follicles, provides the sentinel barrier protecting the body from dehydration, injury, and infection. However, these functions of the skin are compromised when encountering exogenous trauma such as burn injury or endogenous disrupting factors, such as a lack in blood supply. For recovery from small wounds the intrinsic healing capability of the skin is quite sufficient, even more so when assisted with conventional medical therapy. However, any deeper injury extending to the dermis and beyond requires surgical intervention and usually the area of the defect must be covered either by transplanted skin or by an artificial skin replacement (Lagus&Vuola, 2004) after the evacuation of any underlying damaged tissues. The ideal covering is a meshed skin autograft taken from a healthy area with a preferable color match to the site of injury. When using the patient' s own, autologous, cells and tissues the graft is not rejected, and is accepted as a permanent patch by the body's immune system. The most crucial limiting factor for such autologous transplantation is the lack of available donor sites in extensive burn injuries. Moreover, covering large defects with few donor sites requires extensive graft stretching that leads to poor cell outgrowth and graft integration at the host site.

The treatment of severe skin wounds with cultured keratinocytes, a method already introduced two decades ago in burn medicine, has been considered a promising alternative for therapy in limited availability of autologous split skin grafts (Atiyeh et al., 2007; Chester et al., 2004)). The main methods, transplantation of keratinocytes as single cell suspensions or as cultured epithelial sheets, each have specific problems related. Cultured sheet grafts have been shown to be initially fragile, susceptible to infection, and to require special handling techniques (Compton et al., 1998). Cultivation of keratinocyte sheet grafts requires a lengthy time period of 3 to 4 weeks causing delay in treatment (Rouabhia, 1996). Upon transplantation to the patient, relatively unpredictable rates of incorporation and coverage ("take") have been observed with transplanted sheets and well as instability of the healed skin (Chester et al., 2004).

Various reports have shown that single cell keratinocyte transplantation therapy combined with fibrin glue (Tisseel®) as carrier matrix hastens reepithelialisation and improves the quality of healed skin (Currie et al., 2001). Clinical trials conducted on fibrin glue as a carrier matrix have shown increased cell take as well as survival of keratinocytes on the wound bed (Vanscheidt et al., 2007). Some authors have, however, noted that fibrin glue may eventually inhibit keratinocyte migration and cell viability may be decreased (Kubo et al., 2001). Factors contributing to decreased motility may lie in migration promoting growth factors such as EGF, which has been shown to enhance cell migration on a fibrin matrix (Pellegrini et al., 2009).

Mesenchymal-epithelial interactions in the skin between fibroblasts and epithelial keratinocytes present a complex interplay consisting of various cytokines and growth factors guiding cell proliferation, migration as well as differentiation. This interplay is equally crucial in mesenchymal-epithelial interplay taking part in skin substitutes (Spiekstra et al., 2007). As seen with Rheinwald and Greens method of cultivating keratinocytes on a feeder layer, mesenchymal paracrine signals, such as growth factors and cytokines, remain essential for keratinocyte proliferation and survival (Werner et al., 2007). One versatile dermis derived growth factor implicated in tissue regeneration and wound healing is hepatocyte growth factor/scatter factor (HGF) (Conway et al., 2006). It has been linked to increased migration and proliferation of epithelial cells and its ligand- specific receptor c-Met is expressed in keratinocytes (Chmieloviec et al., 2007).

We previously showed that mesenchymal cell-cell activation in nemosis produces massive amounts of HGF (Bizik et al., 2004), and that stimulation of keratinocytes by nemosis- derived factors enhances their migration utilizing the HGF/c-Met/PI3K pathway (Peura et al. 2009 submitted manuscript). Nemosis is a novel biological programmed cell response to high-density cell-cell contacts, and can be effectively activated in dermal fibroblasts (Kankuri et al., 2005; Kankuri et al., 2008). In a therapeutic context, however, application of soluble mediators for topical treatment requires a carrier matrix.

Burn injury is defined as tissue damage resulting from exposure to heat, chemicals, elec- tricky, sunlight, or nuclear radiation. Burn injuries with damage to the deeper layers of the skin, dermis, and to the underlying tissues require skin grafting. Facilitating skin graft growth and coverage of the defect area would decrease the patients' propensity for requiring infections and also to decrease the time required for hospitalizations thus leading to marked improvements in therapy as well as to savings in treatment costs.

Chronic ulcers, such as (i) venous ulcers, (ii) diabetic ulcers, and (iii) pressure ulcers, represent a significant economic burden, including several millions of nonworking days in the United States alone. In terms of diabetic complications, chronic foot ulcerations constitutes up to 50% of complications associated with diabetic care (Medina et al., 2005). These wounds are highly disabling and require constant medical care with frequent hospitalizations and amputations. The wounds' inability to heal properly is due to insufficient blood flow and supply of nutrients to support cell growth combined with increased bacterial growth and inability of the wound to contract at the site of initial trauma (Medina et al., 2005; Mustoe et al., 2006). Based on this rationale, chronic wounds represent a lucrative target for enhanced cell transplantation therapies.

Of crucial hindrance for a more generalized usage of cultured epithelial autografts is the relatively long time needed for purification of cultures and cell stimulation to achieve a relevant area for replacement. This usually takes 3 to 4 weeks (Chester et al., 2004) during which time the patient's wounds are left susceptible for bacterial inoculation and growth leading at worst to the development of sepsis and septic shock.

In this study we show that, mimicking a dermal component, an active matrix incorporating factors released from nemosis-activated fibroblasts is superior to a "blank" matrix or a ma- trix incorporating recombinant HGF in terms of their ability to trap and maintain viability of keratinocytes transplanted on top of it. It is thus shown that fibrin glue could trap the factors released from nemosis, and that by this manner the effects of this carrier matrix may be enhanced to support keratinocyte adherence and proliferation. Such a matrix could then be transplanted with keratinocytes to the site of injury, and would contain an active, growth factor-releasing, dermal component.

We have thus found novel means to promote autologous graft-based therapy of skin wounds using cell and growth factor therapy as released from active wound dressings.

We show here that in vitro conditions a construct formed from fibrin glue incorporated with nemotic signals such as HGF efficiently stimulates keratinocyte migration and proliferation on this fibrin matrix. We also show that this cell matrix supports primary keratino- cyte adherence and proliferation as well as enhances cell viability. We propose that nemo- sis derived stimulus could be utilized in skin wound healing combined with a fibrin matrix to improve cell take as well as migration and combined with transplantation of keratinocyte single cell suspension this matrix would support keratinocyte take, viability and migration to the wound bed, thus hastening epithelialisation and wound coverage.

A regenerative active matrix (RAM) is described that will enable incorporation and delivery of cells activated by nemosis. The matrix will be described not only for its cell growth, scattering, graft integration, and angiogenesis-inducing properties, but also in terms of safety to the patients and efficacy of nemosis cell therapy delivery. Our investiga- tion involves the use of autologous fibroblasts, mesenchymal stromal cells from bone marrow or fat combined with the use of two extracellular matrices i) a biodegradable recombinant collagen matrix constituted of type I or III collagen fibers and/or ii) a non-degradable cellulose matrix. The cells incorporated within the matrix are activated by a novel innovation as initially described by us showing massive activation of mesenchymal cells to pro- duce growth factors when cultured in a specific three dimensional setting (Bizik et ah, 2004, Kankuri et al, 2005).

It should be noted that a regenerative active matrix which contains secreted factors, i.e. mainly growth factors which such activated cells secrete into the growth medium, is an important embodiment of the invention. In a further embodiment of the invention it is also possible to add both said cells and said secreted factors into the matrix. In such a "triple" approach the cell component may be added as spheroids (multicellular clusters) or as individual cells. Various cells may be used in this approach, e.g. adult stem cells, non-human embryonal stem cells, mesenchymal cells (myoblasts, fibroblasts, etc.) or epithelial cells (keratinocytes etc.).

In one clinical indication the product of the invention may be applied in a carrier matrix to a surgical wound caused when a skin graft is taken for transplantation. It may also be used to stimulate skin graft integration to burn injuries, and healing of acute wounds. In general, any kind of wounds may be treated, e.g. burn injuries, acute wounds, chronic wounds, including chronic foot ulcerations and chronic skin wounds, large wounds, donor site wounds, surgical wounds, mucosal wounds, venous ulcers, diabetic ulcers and pressure ulcers.

Advantages of the product in comparison with such products as growth factors manufactured by recombinant technology are i) the composition deriving from activated human cells is biologically optimized and synergistic for initiating human target cell responses for healing, ii) it is manufactured without artificial chemical manipulation and is a cell therapy product rather than a drug, iii) the patient's own cells can be used for its manufacture for individualized responses, and iv) it does not include cells that upon administration and with time could develop into uncontrollably growing tissue. Conventional matrices thus provide a support or slow-release of recombinant substances and drugs, but the active matrix of the present invention provides conventional support but carries cellular bioreactors that produce high concentrations of biological mediators.

The active matrices, cellulose and collagen are used in combination for superior stimulation of transplanted autologous cells as well as the wound bed. Figure 7 shows one alterna- tive of their combined use. The regenerative active matrix is placed on top of the engineered active collagen-keratinocyte layer and releases growth factors, such as HGF/SF, to the underlying structures.

By its high absorbing capacity it also drains wound exudate as well as bacteria from the wound, thus presenting a dual action for this component. The cellulose matrix can alternatively be used as a supporting structure for the collagen matrix. The collagen matrix is mixed with autologous cells induced to undergo nemosis. Such an active collagen matrix acts as a dermal equivalent to stimulate adherence and growth of keratinocytes as well as promoting graft integration at the host site.

A combination of collagen and cellulose incorporating the nemosis-activated cells can be used instead of cellulose on top of grafted cells or split- thickness meshed autograft. Such a hybrid matrix combines beneficial components of both innovations synergizing to bring support and added maneuverability for the gel-like structure of collagen as such. On top of a meshed autograft this replenishable and replaceable active hybrid matrix provides long term delivery of nemosis-based cell therapy to the graft leading to increased keratinocyte outgrowth from the borders of the intricities of the meshed graft. This stimulatory approach enables faster coverage of the wounded area with autologous keratinocytes even when the graft need to be extensively expanded (Figure 8).

We thus describe designing of a novel working concept of an active collagen/cellulose matrix for the purpose of targeted cell-based therapy and for on-site release of growth factors such as HGF/SF. While collagen/cellulose matrices have advantageous properties, fibrin matrices may be also be used. A further embodiment is using a cell sheet, which comprises cell-cell-interactions, or interactions between cells and an extracellular matrix.

Brief Description of Drawings

Figure 1 (a) Fibrin matrix incorporating fibroblast nemosis signals supports keratinocyte proliferation. Equal amount of green fluorescent protein-labelled HaCaT cells were seeded on top of fibrin matrix formed from nemosis or monolayer conditioned medium. One day after adherence of the cells, the matrix was washed with PBS and serum deprived. Fluorescence of the cells was measured 4 days after seeding. Data expressed as mean + SEM from three independent experiments (n=4). *** p<0.001, as compared to control matrix. Figure 1 (b) Fibrin matrix with recombinant HGF supports keratinocyte proliferation. HGF-induced proliferation was inhibited with specific c-Met kinase-inhibitors SUl 1275 (85OuM) and PHA66574. *** p<0.001, * p<0.05 as compared to vehicle-treated cells. Figure 1 (c) Fibrin matrix embedding nemosis supports primary keratinocyte viability. Primary keratinocytes obtained from patients were seeded on fibrin matrix containing nemo tic or monolayer conditioned medium. Data expressed as mean + SEM from two inde- pendent experiments (n=3), ** p<0.01 as compared to control.

Figure 2 (a) Fibrin matrix embedding nemosis supports primary keratinocyte viability. Primary keratinocyte viability (MTT) on Tisseel - 4^th day after seeding. Figure 2 (b) Fibrin matrix with recombinant HGF supports keratinocyte proliferation. HGF-induced proliferation was inhibited with specific c-Met kinase-inhibitors SUl 1275 (85OuM) and PHA66574.Day 4 after seeding of cells on fibrin lattice.

Figure 3 (a) and (b) Amount of cells attached to fibrin lattice (a) seeding of keratinocytes, (b) seeding of conditioned medium .

Figure 4 (a) Seeding of GFP-labeled HaCaT cells on fibrin matrix (Tisseel, Baxter) incorporated with nemosis/monolayer conditioned medium.

Figure 4 (b) Inhibition of HGF-induced GFP-labeled HaCaT attachment on fibrin matrix (Tisseel).

Figure 5 (a) and (b) Keratinocyte migration on fibrin matrix (Tisseel Duo Quick, Baxter) (a) HaCaT stimulation with monolayer conditioned medium, (b) HaCaT stimulation with nemosis conditioned medium. Figure 6 A schematic representation of skin histology. Cells of the epithelial or epidermal layer are mitotically active in the basal layer anchored to the basement membrane. As cells mature they migrate upward and form spinous processes, flatten to lose their column-like appearance, and adhere more tightly to each other forming eventually the outer stress- resilient layer of the skin. The mesenchymal supporting connective tissue of the dermis is produced mainly by the dermal fibroblasts. Figure adapted from Alonso&Fuchs (2003).

Figure 7 Schematic reconstruction of active cellular matrix use for stimulation of both autologous keratinocytes cultured on collagen and wound bed. The regenerative active matrix (RAM) can incorporate keratinocytes, fibroblasts, or multicellular spheroids and promote their growth and differentiation.

Figure 8 Schematic reconstruction of active hybrid matrix on top of an autologous kerati- nocyte graft or meshed autograft. The collagen component adsorbs HGF/SF and matrix contacts for cells whereas the cellulose component adds strength to the collagen matrix structure. The hybrid active matrix is readily replaceable and removable at any time.

Experimental

Materials and Methods

Cell cultures

Spontaneously immortalized keratinocytes (HaCaT) were a kind gift from Dr Norbert Fusenig (Heidelberg University, Heidelberg, Germany). The cells were grown in DMEM supplied with 1% penicillin/streptomycin, 5% fetal calf serum (FCS) (Sigma- Aldrich).

Isolation and culture of keratinocytes

Biopsy specimens of skin from surgical waste were transferred to the laboratory in gauze soaked in physiological saline. Keratinocytes were isolated within 24 h and the tissue was kept in +8 ⁰C until use. After washing with phosphate buffered saline (PBS) subcutaneous fat was removed and approximately lmm wide skin pieces containing epidermis and dermis were cut for further processing. Epidermal de-attachment was performed with over- night incubation in Dispase® (1.9U/ml). After incubation the epidermis was lifted peeled from the dermis and transferred to 0.1% trypsin. A single cell suspension was obtained with mechanical dissociation with suspending the epidermal sheets with a pipette. Keratinocytes were grown in defined keratinocyte serum free medium K-SFM (Invitrogen, Life Technologies Corporation, Carlsbad, CA) supplied with 1% penicillin/streptomycin. Cells were seeded on 24- well plates for measurement of wound healing. Cells were onto pre-performed fibrin lattice for measurement of cell viability.

Reagents The following primary antibodies were used for immunoblotting: rabbit monoclonal antibody against c-Met (Neomarkers, Fremont, CA), rabbit monoclonal anti-phospho-Met (Y1234, Y1235) (Upstate Biotechnology, Lake Placid, NY), mouse monoclonal antibody against actin (NeoMarkers). Recombinant human HGF (rhHGF) was from PeproTech, Inc (Rocky Hill, NJ). A selective small molecule Met-kinase inhibitor SUl 1274 was used at indicated concentrations to show HGF/c-Met dependency and another c-Met inhibitor PHA 665752 was from Sigma-Aldrich and used at indicated concentrations. Tisseel® Duo Quick (Baxter) was used for formation of fibrin matrix.

Measurement of cell viability Cell respiration, an indicator of cell viability, was assayed by the mitochondria-dependent reduction of 3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to for- mazan (Roche). At selected time points, the cultures were incubated with the substrate (5 mg/ml) for 90 min for primary keratinocytes and 60min for HaCaT cells at 37.1 ⁰C; then the medium was aspirated and MTT was dissolved in DMSO.

The extent of MTT conversion to formazan was quantified by measurement of optical density at 540 nm with a wavelength correction of 620 nm, using a microplate reader (Multi- scan MCC/340, Labsystems, Helsinki, Finland).

Initiation of nemosis

Multicellular spheroids were formed as described previously. Briefly, U-bottomed 96-well plates (Costar, Cambridge MA) were treated with low-electroendosmotic agarose (Bio Whittaker, Rockland, ME) prepared in sterile water to form a thin film of nonadhesive sur- face. CRL-2088 fibroblasts were seeded 15000 cells per spheroid in 80μl DMEM with 10% FCS. Spheroid formation was allowed to proceed for one day and after this they were collected, let to settle spontaneously by gravity, and were washed with serum-free medium. Spheroids were then collected and seeded in serum free medium for incubation of medium. As a control, same amount of BMSCs were directly grown as monolayer culture in 10% FCS-DMEM for one day and then cells were washed and serum was deprived. Conditioned medium from spheroids as well as the monolayers were collected 5 days after incubation. Medium was dialyzed (Slide-A-Lyzer), fully lyophilized and the remaining protein fraction was replaced with either DMEM or KSF-m.

Fibrin lattice

The fibrin matrix was formed using commercially available fibrin glue (Tisseel® Duo Quick, Baxter). For optimization of keratinocyte adherence to fibrin matrix both components were diluted for optimization of cell growth and proliferation. The fibrin gels were prepared as follows: the original thrombin stock solution of 500IU/ml was diluted to a final concentration of 3 IU/ml and the fibrin component from 95mg/ml to 7.5 mg/ml. The diluted solutions were mixed in the bottom of a 24- well plate and were kept in 4⁰C for Ih to form stable gels.

Measurement of cell proliferation on fibrin lattice

With HaCaT cells their proliferation on fibrin lattice was analyzed by measuring fluorescence signal from GFP-labeled HaCaT cells. The cells were labelled with green fluorescent protein (GFP) by incubation with a lentiviral vector carrying the gfp gene and 8 μg/ml polyprene for 24 hours. The lentiviral vector was a kind gift from professor Seppo YIa- Herttuala (AIV Institute, Kuopio, Finland). Seeding of cells onto lattice as made FCS 5% and medium was changed after 6 hours. At this point viable cells had adhered onto lattice. Cell proliferation was measured at indicated time points.

Primary keratinocytes were seeded on top of fibrin sheets prepared in KSF-m medium with or without nemotic conditioned medium. Cell viability was measured with MTT on indicated time points.

The entire contents of all the references cited herein are hereby incorporated by reference. References

Alonso L, Fuchs E. Stem cells of the skin epithelium. Proc Natl Acad Sci U SA , 2003; 100:11830-11835.

Atiyeh BS, Costagliola M. Cultured epithelial autograft (CEA) in burn treatment: three decades later. Burns. 2007; 33:405-13

Bizik J, Kankuri E, Ristimaki A, Taϊeb A, Vapaatalo H, Lubitz W, Vaheri A. Cell-cell con- tacts trigger programmed necrosis and cyclooxygenase-2 expression. Cell Death Differ 2004; 11: 183-95

Chester DL, Balderson DS, Papini RP. A review of keratinocyte delivery to the wound bed. J Burn Care Rehabil. 2004; 25:266-275

Chmielowiec J, Borowiak M, Morkel M, Stradal T, Munz B, Werner S, Wehland J, Birchmeier C, Birchmeier W. c-Met is essential for wound healing in the skin. / Cell Biol 2007; 177: 151-62 Compton CC, Butler CE, Yannas IV, Warland G, Orgill DP. Organized skin structure is regenerated in vivo from collagen-GAG matrices seeded with autologous keratinocytes. / Invest Dermatol 1998;110:908-16

Conway K, Price C, Harding K, Jiang W. The molecular and clinical impact of hepatocyte growth factor, its receptor, activators, and inhibitors in wound healing. Wound Repair Re- gener 2006; 14: 2-10

Currie LJ, Sharpe JR, Martin R The use of fibrin glue in skin grafts and tissue-engineered skin replacements: a review. Plast Reconstr Surg. 2001; 108:1713-26

Kankuri E, Cholujova D, Comajova M, Vaheri A, Bizik J. Induction of Hepatocyte growth factor/scatter factor by fibroblast clustering directly promotes tumor cell invasiveness. Cancer Res 2005; 65: 9914-22 Kankuri E, Babusikova O, Hlubinova K, Salmenpera P, Boccaccio C, Lubitz W, Harjula A, Bizik J. Fibroblast nemosis arrests growth and induces differentiation of human leukemia cells. Int J Cancer. 2008; 122: 1243-52

Kubo M, Van de Water L, Plantefaber LC, Mosesson MW, Simon M, Tonnesen MG, Taichman L, Clark RA. Fibrinogen and fibrin are anti-adhesive for keratinocytes: a mechanism for fibrin eschar slough during wound repair. J Invest Dermatol. 2001; 117:1369-81

Lagus H, Vuola J. Artificial skin equivalents. Duodecim. 2004; 120:1977-1985. Finnish

Medina A et al. Pathophysiology of chronic nonhealing wounds. J Burn Care Rehabil. 2005; 26:306-319. Mustoe TA et al. Chronic wound pathogenesis and current treatment strategies: a unifying hypothesis. Plast Reconstr Surg. 2006; 117:35S-41S.

Pellegrini G, Rama P, Mavilio F, De Luca M. Epithelial stem cells in corneal regeneration and epidermal gene therapy. J Pathol. 2009 Jan; 217:217-28

Rouabhia M. Permanent skin replacement using chimeric epithelial cultured sheets comprising xenogeneic and syngeneic keratinocytes. Transplantation. 1996; 61:1290-300 Spiekstra SW, Breetveld M, Rustemeyer T, Scheper RJ, Gibbs S. Wound-healing factors secreted by epidermal keratinocytes and dermal fibroblasts in skin substitutes. Wound Repair Re gen. 2007; 15:708-17

Vanscheidt W, Ukat A, Horak V, Bruning H, Hunyadi J, Pavlicek R, Emter M, Hartmann A, Bende J, Zwingers T, Ermuth T, Eberhardt R. Treatment of recalcitrant venous leg ulcers with autologous keratinocytes in fibrin sealant: a multinational randomized controlled clinical trial. Wound Repair Re gen. 2007; 15: 308-15

Werner S, Krieg T, Smola H. Keratinocyte-Fibroblast Interactions in Wound Healing. / Invest Dermatol 2007; 127: 998-1008

Claims

1. A pharmaceutical composition comprising an active carrier matrix into which cells activated by nemosis or - factors released from nemosis-activated cells or a combination of said cells and said secreted factors have been incorporated.

2. The pharmaceutical composition according to claim 1, wherein the carrier matrix is se- lected from the group consisting of fibrin glue, cellulose, collagen and mixtures thereof.

3. The pharmaceutical composition according to claim 1, wherein the nemosis-activated cells are nemosis-activated fibroblasts.

4. The pharmaceutical composition according to claim 3, wherein the factors released from nemosis-activated fibroblasts are growth factors secreted by mesenchymal cells when cultured under conditions in which said cells form multicellular spheroids.

5. The pharmaceutical composition according to claim 3, wherein a solution of the secreted growth factors is in the form of a lyophilizate.

6. The pharmaceutical composition according to claim 1, wherein in the combination of said cells and said secreted factors the cells are selected from adult stem cells, non-human embryonic stem cells, mesenchymal cells and epithelial cells.

7. The pharmaceutical composition according to claim 6, wherein the mesenchymal cells are selected from myoblasts and fibroblasts.

8. The pharmaceutical composition according to claim 6, wherein the epithelial cells are keratinocytes.

9. A composition comprising an active carrier matrix into which cells activated by nemosis or factors released from nemosis-activated cells or a combination of said cells and said secreted factors have been incorporated, for use in a method for treating a wound.

10. The composition according to claim 9, wherein the wound is selected from the group consisting of burn injuries, acute wounds, chronic wounds, including chronic foot ulcerations and chronic skin wounds, large wounds, donor site wounds, surgical wounds, mucosal wounds, venous ulcers, diabetic ulcers and pressure ulcers.